This invention pertains to uses of stimulated whole blood collection devices and their utilities related to virologic infections, viral vector systems and viral treatment regimens, including latent cellular reservoirs and drug resistance.
Despite successes of highly active antiretroviral therapy (HAART) inhibiting Human Immunodeficiency Virus (HIV) replication, viral latency and low-level replication enable viral persistence and prevent eradication. Achievement of either long-term HIV control in the absence of ART (functional cure) or complete elimination of HIV from the human body (sterilizing cure) remain unmet challenges (2, 3). Although a variety of cell types such as macrophages and long-lived resting memory CD4+ T cells are known viral reservoirs (7, 15), these and other latent HIV reservoirs are difficult to investigate owing to sampling volumes and limited availability of ex vivo human tissues. Understanding molecular mechanisms of HIV-1 latency in vivo and the dynamics of the latent reservoir is also complicated by the small numbers of latently infected cells and the lack of known phenotypic markers distinguishing them from uninfected ones.
Recently, the size of the latently infected virus reservoir in resting memory CD4+ T cells has been determined to be 60-fold greater than previously estimated [Y-C Ho 2013]. Current methods of T-cell activation reverse less than 1% of provirus to release infectious virus. Methods and therapeutic treatments to clear provirus are urgently needed to provide either functional or sterilizing curative strategies. The present invention provides a method for testing such strategies using whole blood culture to measure virus production, provirus, activation and inhibition of virus production.
Current knowledge of the latent viral state is largely based upon limited animal models (4, 5, 11, 16) and multiple T cell models (17) that can be implemented in laboratory settings. Such models allow investigators to manipulate conditions and clinical parameters otherwise difficult to control for in humans. Leukophoresis; sucrose or ficoll gradients; and antibody affinity/magnetic beads techniques are currently employed for the isolation of primary white blood cells from donors. Such processes may disturb the natural state of recovered cells and remove (or add) critical factors altering their normal function. While useful, none of these experimental systems sufficiently replicates biological properties of HIV infection, residing in multiple cell types and established through complex mechanisms. Therefore, to support goals of discovering and developing methods to purge the latent reservoir, and for identifying useful therapeutic patient strategies there is a critical need for improved models useful for determining the source and dynamics of persistent virus, for conducting sampling of specimens from naturally infected individuals, for characterizing activation of latent reservoirs and for identifying useful markers corresponding to latently infected T cells. Incremental changes in reservoir size and activity need to be consistently detected in order to identify promising therapies in a cost-effective and safe manner, reducing exposures to individuals. Novel and improved assays are also required to provide reproducible, sensitive, precise, feasible, accessible and standardizable measurements of persistent replication-competent virus, HIV DNA genomes, and tissue reservoirs of HIV in support of large clinical trials, for research purposes and for personalized medicine. Stimulated whole blood culture provides commercially available products to noninvasively evaluate the                extent of viral replication        cellular sources of virus production        insult to the immune system        extant viral quasispecies        activation process of latently infected cellular reservoirs        effectiveness of new curative strategies        personalized approaches for patient treatment        effects of virus, bacteria or fungal coinfections        cotherapeutic complicationsDrug Resistance        
Current estimates report 34.0 million people are living with HIV globally; 2.5 million people were newly infected in 2011 (1). In the United States, 1.1 million people are estimated to be living with HIV; approximately 50,000 people are newly infected each year. Approximately 18% of U.S. HIV-infected individuals are unaware of their infection status; some evidence indicates that ˜half of new infections occur between 15-24 years of age (2).
Although antiretroviral therapeutics (ARTs) have greatly extended lifespans, new challenges related to long-term management of HIV have arisen. Daily adherence (>95%) to multiple therapeutics is mandatory to control disease progression (3, 4). FDA-approved ART drugs belong to limited drug classes. HIV produces very large numbers of mutational variants owing to high rates of virus particle production (109-1010 per day, unsuppressed), careless genomic proof-reading (reverse transcriptase) and selective drug pressure (5). Despite years of continuous successful ART suppression, viral reservoirs lurking in cells and physiological compartments threaten to reestablish active infection (6), typically within ˜9 to days upon ART withdrawal or treatment interruption (7). Resurgence of virus reflects an archived historical collection of lifelong mutational variants, including their ART resistance profiles (6, 8-11). As HIV-infected individuals are infected young and living longer, ART options for retaining antiviral activity dwindle. As such, ART resistance testing has become a standard of care in HIV infection management.
HIV is now largely considered a chronic disease. Consequently, drug resistance in the face of limited drug classes is a mounting problem (12) leading to treatment failure with virus production surges and associated health risks. Furthermore, in the U.S. it is reported that approximately 6-16% of newly diagnosed HIV-infected individuals are now initially resistant to at least one ART class of drugs at first line therapy due to transmitted drug resistant mutations, consequently increasing risks for treatment failure (13-19). The extent of drug resistance further correlates with duration of uncontrolled virological replication (20), and clinically presents as increasing plasma virus production (viremia), decreasing CD4+ T-cell counts and the onset of both AIDS and non-AIDS related conditions (21, 22). Poor control of HIV is associated with higher costs (23, 24). The goal of ART is to preserve T-cell counts (>350 counts/uL) and activity through control of viremia (<50 viral RNA copies/mL) (25). There is currently no consensus on best treatment strategies for third-line treatment failure (“salvage therapy”); management of these patients is thus extremely challenging (26-29).
Since maintaining low to undetectable viremia (<50 copies/mL) with ART is the key to surviving HIV, detecting and quantifying drug resistance is critical for therapeutic selections (30, 31). The US Department of Health and Human Services (DHHS) guidelines for ART (25) state:
‘Selection of a regimen should be individualized on the basis of virologic efficacy, toxicity, pill burden, dosing frequency, drug-drug interaction potential, resistance testing results, and comorbid conditions, and that based on individual patient characteristics and needs, in some instances, an alternative regimen may actually be a preferred regimen for a patient.’
Currently available FDA-approved resistance testing includes two genetic sequencing (genotyping) methods for detecting HIV genomic mutations. Alternative unapproved in vitro drug response (phenotyping) systems and other genotyping systems are also available to meet recommendations by the DHHS. Unfortunately, due to a lack of overall sensitivity, existing systems fail to adequately characterize viral load and mutational variants in that they require samples expressing HIV plasma viremia levels >500-1000 RNA copies/mL. Furthermore, the complexity of these detection platforms often necessitates the construction of analysis clones, recombinant vector systems, amplification of isolates or sequence-specific probes (32, 33). Current generation tools are lacking in overall sensitivity and specificity leading to as many much as 10-25% of minority virus quasispecies being overlooked. Test results also often require several weeks and rely upon complex assays performed by highly skilled staff using expensive instruments. Their utilization of historical data coupled with proprietary algorithms for interpretation sometimes lead to agreement failures (34-36). Furthermore, FDA-approved (2002-2003) genotyping is limited to (N)NRTI (non/nucleoside reverse transcriptase inhibitors) and PI (protease inhibitors) drug classes of HIV-1 subtype B, with integrase inhibitor resistance testing performed separately. For complex patterns of resistance, genotyping sequence comparisons cannot adequately predict responses (35, 37), for which time-consuming phenotyping assays are more appropriate. Expanding drug classes, emerging patterns of resistance and evolving virus strains (including non-B subtypes) argue for technological adaptation.